For the DNA content, frozen samples were weighed and DNA extracted and purified with the DNeasy kit (Qiagen Inc., Valencia, CA). Total DNA content was determined using the PicoGreen dsDNA assay (Invitrogen). For GAG quantification, minced samples were lyophilized for 24 hours and digested overnight at 60°C in 125 µg/mL papain type III (Sigma-Aldrich) in phosphate buffer with EDTA pH 6.5 (100 mM disodium phosphate, 10 mM ethylenediaminetetraacetic acid, and 10 mM l -cysteine). GAG content was determined spectrophotometrically using the Blyscan Glycosaminoglycan Assay Kit (Biocolor Ltd., Carrickfergus, UK). The GAG and DNA content in engineered cartilage was expressed as % of native GAG and % of native GAG/DNA ratio. The GAG content averaged 118.4 ± 40.5 and 141.2 ± 25.3 µg/mg dry tissue for sheep and human native auricular cartilage, respectively. GAG/DNA ratio was 1.0 ± 0.2 for both species.
Papain type 3
Manufactured by Merck Group
Sourced in United States
Papain type III is a proteolytic enzyme derived from the papaya plant. It is used as a general-purpose laboratory reagent for the digestion and hydrolysis of proteins. The core function of Papain type III is to facilitate the breakdown of peptide bonds in proteins, making it a useful tool for various biochemical and analytical applications.
Automatically generated - may contain errors
3 protocols using papain type 3
1
Quantification of GAG and DNA in Engineered Cartilage
2
Quantitative Measurement of GAGs
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For quantitative measuring of GAGs, the total GAGs content of cell/polymer constructs was determined using papain digestion and the dimethylmethylene blue dye method. Samples were lyophilized for 24 h and then digested under sterile conditions with papain type III (Sigma) at 125 mg/ml in a buffer of 0.1 M NaH2PO4, 5 mM methylenediaminetetraacetic acid, and 5 mM cysteine hydrochloride at pH 7.0 overnight at 60°C before the dye was added. Dimethylmethylene blue stock solution was made using dimethylmethylene blue, sodium chloride, glycine, sodium aziade in 1 N hydrochloride, and water. A spectrophotometer set at a wavelength of 520 nm was used to measure the optical density of the digested samples. Glycosaminoglycan was measured and reported in micrograms per milliliter per milligram of dry weight tissue.
3
Quantifying cartilage sGAG content
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The cartilage tissue that separated from each OCA (n = 4) was freeze-dried and incubated with a papain digestion solution (5 mM L-cysteine, 100 mM Na2HPO4, 5 mM EDTA, and 125 mg/ml papain type III: Sigma-Aldrich, Missouri, USA) overnight at 60°C. At this point, a 1,9-dimethyl- methylene blue (DMMB) Blyscan™ Glycosaminoglycan test kit was used to determine the total sGAG content (b1000, bicolor, UK) followed by manufacturer’s instruction.
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